1. Volume 16, Issue 11, Pages 1158-1168 (November 2009)
Identification and Characterization of a Small Molecule Inhibitor of Formin-Mediated Actin Assembly 
Syed A. Rizvi, Erin M. Neidt, Jiayue Cui, Zach Feiger, Colleen T. Skau, Margaret L. Gardel, Sergey A. Kozmin, David R. Kovar 
Chemistry & Biology 
Volume 16, Issue 11, Pages (November 2009)
DOI: /j.chembiol
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2. Figure 1 Structure and Activity of SMIFH2 Analog Molecules
(A) Structure of SMIFH2 (1) and analog molecules 2–7.
(B) Plot of the dependence of the assembly rate of 2.5 μM Mg-ATP actin monomers (20% pyrene-labeled) in the presence of 25 nM mouse formin mDia1 on the concentration of SMIFH2 (1) (●) and analog molecules 2 (♦), 3 (○), 4 (△), 5 (□), 6 (⋄), and 7 (■). Conditions were the same as in Figure 2.
(C) Fission yeast cells expressing either GFP-CHD (top panels) to label the entire actin cytoskeleton or type V myosin Myo52-GFP (bottom panels), following treatment for 30 min at 25°C with 10 μM of the indicated analog. Numbers in the left corner of lower panels represent the percentage of cells in which Myo52-GFP is localized specifically to cell tips via formin-dependent actin cables.
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3. Figure 2 SMIFH2 Inhibits Formin-Mediated Actin Assembly In Vitro
(A) Time course of the polymerization of 2.5 μM Mg-ATP actin monomers (20% pyrene-labeled) with 2.5 μM profilin MmPRF1 in either the absence (thick curve) or presence of 25 nM mDia1(FH1FH2) and 0.0 (■), 7.5 (●), 10 (♦), or 100 (▴) μM SMIFH2. Conditions: 10 mM imidazole (pH 7.0), 50 mM KCl, 1 mM MgCl2, 1 mM EGTA, 0.5 mM DTT, 0.2 mM ATP, and 90 μM CaCl2.
(B) Plot of the dependence of the maximum polymerization rate of 2.5 μM Mg-ATP actin on the concentration of SMIFH2 in the presence of the indicated formin constructs.
(C) Bar graph of the effect of 50 μM SMIFH2 on the maximum polymerization rate of 2.5 μM actin monomer in the absence (No Formin) or presence of diverse formin (FH1FH2) constructs: 25 nM mouse mDia1, 25 nM mouse mDia2, 100 nM nematode worm CYK-1, 100 nM fission yeast Cdc12, 10 nM fission yeast Fus1, and 75 nM budding yeast Bni1. Error bars, SD; n = 3.
(D) Fluorescent micrographs of the products of actin polymerization assays from (C) stained with rhodamine-phalloidin. Bar, 1.0 μm.
(E and F) Effect of 100 μM SMIFH2 or Arp2/3 complex inhibitors CK-666 and CK-869 (Nolen et al., 2009) on the polymerization of 2.5 μM actin monomers with 25 nM Arp2/3 complex and 100 nM GST-WASP-VCA. Error bars, SD; n = 3.
(G–J) Effect of SMIFH2 on the elongation of filaments preassembled by formin. Unlabeled actin (2.5 μM) was preassembled alone or in the presence of 50 nM Cdc12(FH1FH2) or mDia2(FH1FH2), treated with a range of concentrations of SMIFH2 and diluted 15-fold into new reactions with 0.5 μM Mg-ATP-actin (10% pyrene-labeled) and 5.0 μM profilin.
(G) Time course of the elongation of control filaments preassembled without formin in the absence (○) or presence (●) of 10 μM SMIFH2.
(H) Time course of the elongation of Cdc12-assembled filaments alone (●), with profilin (■), and with profilin and 10 μM SMIFH2 (♦).
(I) Bar graph of the effect of 10 μM SMIFH2 on the maximum elongation rate of control and formin-assembled filaments. Error bars, SD; n = 3.
(J) Plot of the dependence of the polymerization rate on the concentration of SMIFH2 for filaments preassembled by formin.
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4. Figure 3
Evanescent Wave Fluorescent Microscopy of the Effect of SMIFH2 on Spontaneous Actin Assembly
Assembly of 1.0 μM ATP-actin with 0.5 μM Oregon green-labeled ATP-actin in the presence of 5.0 nM mDia1 and 2.5 μM MmPRF1 on slides coated with NEM-myosin II. Conditions: 10 mM imidazole (pH 7.0), 50 mM KCl, 1 mM MgCl2, 1 mM EGTA, 50 mM DTT, 0.2 mM ATP, 50 μM CaCl2, 15 mM glucose, 20 μg/ml catalase, 100 μg/ml glucose oxidase, 0.5% (500 centipoise) methylcellulose, and 1.7% DMSO at 25°C. Bar, 5 μm. Movies of time lapses are published as supplemental data.
(A, C, and E) Time-lapse micrographs of representative 45 × 45 μm areas, with time in seconds indicated at the bottom. Labels indicate control (c) and formin-associated (f) filaments.
(B, D, and F) Plots of the length of six individual filaments versus time for control (solid lines) and formin-associated filaments (dashed lines). The average barbed end elongation rates are indicated.
(A and B) Control reaction without SMIFH2.
(C and D) Reaction with 100 μM SMIFH2.
(E and F) Reaction with 100 μM Analog 2.
(G) Bar graph of the average percentage of formin-nucleated filaments that appear by 450 s in the entire 133 × 133 μm field in both the absence and presence of either 100 μM SMIFH2 or 100 μM Analog 2. Error bars, SD; n = 3.
(H) Dependence of the percentage of formin-nucleated filaments on the concentration of SMIFH2.
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5. Figure 4
Evanescent Wave Fluorescent Microscopy of the Effect of SMIFH2 on Formin-Mediated Actin Assembly From Beads
Assembly of 1.0 μM ATP-actin with 0.5 μM Oregon green-labeled ATP-actin on beads coated with GST-mDia1(FH1FH2) in the presence of 7.5 μM MmPRF1. Conditions were the same as in Figure 3. Movies of time lapses shown in (A) and (C) are published as Supplemental Data.
(A and C) Time-lapse micrographs of representative areas containing three beads (circled), with time in seconds indicated at the bottom. Numbers indicate individual actin filaments assembled from each bead. Bar, 5 μm.
(B and D) Plots of the growth of six individual filament barbed ends versus time for filaments assembled from beads. The average barbed end elongation rate of ten filaments is indicated.
(A and B) Control reaction without SMIFH2.
(C and D) Reaction with 100 μM SMIFH2.
(E) Bar graph of the average number of filaments per bead that appear by 375 s in both the absence and presence of 100 μM SMIFH2. Error bars, SD; n = 3.
(F) Dependence of the average number of filaments per bead that appear by 375 s on the concentration of SMIFH2.
(G and H) Effect of washing beads following treatment with 75 μM SMIFH2.
(G) Bar graph of the average number of filaments per bead that appear by 375 s in reactions without SMIFH2, with SMIFH2, 1 min after SMIFH2 was washed out, and 5 min after SMIFH2 was washed out. Error bars, SD; n = 3.
(H) Representative regions before and after 450 s. The number of filaments polymerized from each bead (circled) are indicated. Bar, 5 μm.
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6. Figure 5
SMIFH2 Inhibits Formin-Mediated Actin Assembly in Fission Yeast
(A and B) Representative micrographs demonstrate that SMIFH2 disrupts the formin-dependent actin cytoskeleton in fission yeast. Scale bars, 5 μm. Fission yeast cells expressing GFP-CHD to label the entire actin cytoskeleton: Arp2/3-dependent actin patches, formin-dependent actin cables (small arrows), and contractile rings (large arrowheads).
(A) Cells treated for 30 min at 25°C with either DMSO, 25 μM actin monomer binding drug Latrunculin A, 25 μM SMIFH2, or 100 μM Arp2/3 complex inhibitor CK-666.
(B) Cells treated with a range of SMIFH2 concentrations for 30 min at 25°C. Top panels show actin cables and bottom panels show contractile rings. Cables are lost with 2.5 μM SMIFH2, whereas contractile rings are present until 25 μM SMIFH2.
(C) Representative micrographs of fission yeast cells expressing various GFP fusions that label specific actin cytoskeleton structures. Cells were treated for 30 min at 25°C with either DMSO or 10 μM SMIFH2. Numbers in the top left corner indicate the percentage of cells containing Arp2/3 complex-dependent actin capping protein patches (Acp2-GFP), type V myosin localized to the cell tip via formin-dependent actin cables (Myo52-GFP) (Win et al., 2001), or normal (not punctate) localization of regulatory light chain (Rlc1-GFP) and type II myosin (Myo2-GFP) to the formin-dependent contractile ring.
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7. Figure 6
Effect of SMIFH2 on Death, Growth, and Migration of NIH 3T3 Fibroblasts
(A) Cytotoxicity of SMIFH2 in 3T3 fibroblasts after 24 hr incubation, determined by measuring ATP content as described in Experimental Procedures. The percentages of viable cells are relative to cells treated with DMSO only. Error bars, SD; n = 3.
(B) Inhibition of the growth of 3T3 fibroblasts by various concentrations of SMIFH2 over 96 hr. Cell viability was measured by ATP content and the luminescence outputs were plotted against incubation time. Error bars, SD; n = 3.
(C) Migration rate of NIH 3T3 fibroblasts treated with DMSO control or 10 μM SMIFH2 for 2 and 6 hr. Error bars, SEM.
(D) Fibroblast morphologies assessed by time-lapse phase-contrast imaging of cells treated with SMIFH2. Lamellipodia (LP) are thin, sheet-like protrusions in spread cells characterized by periods of extension and retraction (indicated by dashed line). Small Bleb phenotype is characterized by phase dark blebs (∼0.5 mm in diameter) that extend and retract along the cell periphery in poorly spread cells (indicated by small arrow). Bulbous phenotype is characterized by poorly spread cells with phase dark regions near the periphery (arrow) and minimal protrusive activity. Large Bleb phenotype is characterized by blebs (>2 mm in diameter) that are static (indicated by large arrowheads). Scale bar, 10 mm. Movies of time lapses are published as Supplemental Data.
(E) Quantification of the percentage of fibroblasts with the indicated cellular morphologies as a function of SMIFH2 concentration.
(F) Distribution of cellular morphologies observed as a function of incubation time for fibroblasts treated with 10 μM SMIFH2 (n > 100 cells for each measurement).
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8. Figure 7
Effect of SMIFH2 on the Actin Cytoskeleton in NIH 3T3 Fibroblasts
(A) Immunofluorescence of filamentous actin and paxillin in NIH 3T3 fibroblasts. Cells that remained spread after SMIFH2 treatment exhibited three distinct actin cytoskeleton organizations: thick F-actin bundles, thin F-actin bundles, and no F-actin bundles. Lamellipodial actin is observed in all phenotypes and indicated by arrows. Scale bar, 10 mm.
(B) F-actin bundles observed in (A) are characterized by their mean width and the maximum filamentous actin intensity normalized to the local background.
(C) Percentage of spread cells exhibiting different actin cytoskeleton phenotypes after 2 hr incubation with indicated concentrations of SMIFH2 (n > 75 cells for each concentration).
Chemistry & Biology  , DOI: ( /j.chembiol )
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